We have begun to develop a detailed description of the metal-catalyzed cleavage (transesterification) of single-stranded RNA through the use of the embedded RNA (embRNA) assay.1-3 EmbRNA contains a single RNA residue in a DNA oligomer. The relative effectiveness of catalysts reported by different groups is often assessed by comparison of these kobs values. We have found that, in a number of cases, the kobs values for cleavage of RNA dimers do not correlate with kobs for cleavage of related RNA oligomers. This makes the dimeric assays potentially misleading in the choice of reagents to cleave high MW, biological RNA samples. We are investigating which factors account for this behavior. Possible contributors include the multitude of metal binding sites on high MW RNA (at phosphodiester, O-4'-ether, 2'-hydroxyl and nucleobase sites), the potential for more than one kinetically-important metal ion, the greater variety of conformations available to oligomeric RNA vs . di meric substrates, and polyelectrolyte effects. Furthermore, as part of our kinetic investigations of RNA cleavage, we have determined the effect of catalyst concentration on kobs for several catalysts, including Ce(III) and La(III) ions and Ce(III) macrocycles. We found that, for aqueous Ce(III), the order of the reaction with respect to Ce(III) varied dramatically. Thus, small changes in catalyst concentration can have huge effects on the observed rate constant. In contrast, the transesterification of embRNA by Ce(III) macrocycles was found to be first order with respect to catalyst.4 This difference is likely due to the suppression of polynuclear species by the macrocyclic ligand. We have determined pH-rate profiles and reaction orders for both "free" Ln3+ ions and the macrocycles, and we are now able to compare these reagents in a meaningful way to Cu(II)- and Zn(II)-based RNA transesterification catalysts. The pH-rate profile helps determine which protonation state of the ca talyst is kinetically important. We have also begun to use multiply-chimeric embRNA substrates (i.e. DNA/RNA/methyl-phosphonate combinations) to determine the role of specific phosphate residues in catalyst binding. Mass spectrometry is required for the identification of cleavage products